[Background] Avian β-defensin 6, an antimicrobial peptide secreted by avian, plays an important role in the resistance to pathogen invasion and immune regulation. However, its conventional expression is inefficient and difficult to be applied in industrial production. [Objective] In order to establish a stable expression of AvBD6 cell line, and detect the antibacterial activity of AvBD6 expression products against drug-resistant E. coli, it can provide reference for the expression of other defensive hormones. [Methods] Observe the transfection efficiency of the constructed eukaryotic recombinant expression vector pLOV-eGFP-AvBD6 after transfection into 293T cells using an inverted microscope; Collect supernatant of 293T cells and infect DF-1 cells, and select stable expression strains by puromycin pressure; Use RT-PCR and Western Blot to detect the expression of the target gene at the transcription level and protein level; Use scanning electron microscopy to observe the antibacterial effect of the cell culture supernatant on drug-resistant E. coli and the damage to the bacteria. [Results] The recombinant expression vector pLOV-EGFP-AvBD6 was successfully constructed, and DF-1 cell lines expressing AvBD6 were screened out. The target gene was expressed at both transcription and protein levels. Cell culture supernatant not only significantly reduced the survival rate of Escherichia coli and Salmonella paratyphi, but low antimicrobial activity against Staphylococcus aureus. [Conclusion] DF-1 cell lines with stable expression of AvBD6 were successfully established. The expression products of DF-1 cells have significant antibacterial effects on drug- resistant E. coli, and have certain guiding significance for the expression and industrial application of defensins.